Dry phase test strips incorporating enzyme-based compositions are used extensively in clinical laboratories, physicians' offices, hospitals and in homes to assay urine or other liquid test samples for analytes such as glucose, cholesterol, lower alcohols and occult blood. The analyte test devices that measure fluctuations in a person's blood sugar, or glucose, levels are especially useful. In fact, these devices have become an every day necessity for many of the nation's several million diabetics Since diabetes can cause dangerous anomalies in blood chemistry and is believed to be a contributor to vision loss and kidney failure, most diabetics must test themselves periodically, then adjust their glucose count accordingly, usually with insulin injections. Patients who are insulin dependent, or about 10% to 15% of diabetics, must test their blood sugar levels as often as four times daily.
Therefore, the detection and quantitative determination of glucose in urine or serum is especially important for diabetic individuals who must control their diet in order to regulate sugar intake and who must be guided in this regard by frequent tests for blood and urine glucose. In addition, screening programs for diabetes depend on the availability of rapid, inexpensive and accurate methods of glucose determination.
Glucose is the sugar most commonly found in urine, although other sugars, such as lactose, fructose, galactose, and pentose, also can be found in the urine under certain conditions. The presence of detectable amounts of glucose in urine is known as glycosuria. Glycosuria occurs whenever the blood glucose level exceeds the reabsorption capacity, or renal threshold, of the renal tubules, that is, when the glomerular filtrate contains more glucose than the tubules are able to reabsorb. Since detectable amounts of glucose in the urine can be related to either a benign or a pathological condition, the physician must distinguish between these two possible conditions and, if necessary, begin medical treatment.
For example, renal glycosuria is observed even though blood glucose levels are normal because if the tubular reabsorption of glucose is below normal, some glucose will spill into the urine. This is a benign condition, as is the occurrence of glycosuria either after eating a heavy meal or during periods of emotional stress. In contrast, diabetes mellitus, the chief cause of glycosuria, is a pathological condition. This pathological condition leads to a noticeable elevation of blood glucose and, usually, to an increase in urine volume. Therefore, in clinical assays, the glucose content of the urine of a diabetic can reach levels as high as 10%, however, values of between 2% and 5% are more common.
As a result, in order to determine if an individual excretes an excess amount of glucose, and in order to monitor the effectiveness of a particular medical treatment, simple, accurate and inexpensive glucose detection assays have been developed. Furthermore, of the several different assay methods developed for the detection or measurement of glucose in urine and serum, the methods based on an enzymatic interaction between glucose oxidase and glucose have proven especially useful.
Testing for blood glucose at home became possible when reagent impregnated test strips for whole blood testing were developed. The reagent test strip includes a reagent composition comprising an enzyme, such as glucose oxidase, capable of catalyzing the oxidation reaction of glucose to gluconic acid and hydrogen peroxide; an oxidizable chromogenic indicator, such as an oxidizable dye, like o-tolidine; and a substance having peroxidative activity capable of catalyzing the oxidation of the indicator. The oxidizable dye or indicator turns a visually different shade of color depending upon the extent of oxidation. The extent of oxidation is in turn dependent upon the concentration of glucose in the blood sample.
The reactions occurring between the glucose and the reagent composition are represented as follows: ##STR1## The reagent test strips generally include a carrier matrix such as a bibulous, e.g., cellulosic, material impregnated with the reagent composition capable of interacting with the glucose as described above.
For several years, diabetics have relied on commercially available urinalysis kits that, despite repeated improvements, have provided imprecise measurements of blood glucose. More recently, reagent test strips to test for glucose in urine were developed. Examples of early urine tests for glucose are described in U.S. Pat. Nos. 2,387,244 and 3,164,534. The most frequently used home urine assays for glucose are either the enzymatic assays based upon the interaction of glucose oxidase and glucose as discussed above or the reduction assays based upon the reduction of certain metal ions by glucose. The enzymatic glucose oxidase assay for glucose, as applied to urine, is specific for glucose. In this assay, like the blood assay, glucose oxidase catalyzes the oxidation of glucose to gluconic acid and hydrogen peroxide. The hydrogen peroxide, in the presence of peroxidase, oxidizes an indicator to produce a color change. Other sugars, such as lactose, fructose, galactose, and pentose are not substrates for glucose oxidase and, therefore, do not interact to cause an eventual color transition in the oxidizable indicator.
The urine glucose assay based on the reduction of metal ions, such as the cupric ion (Cu.sup.++), is nonspecific for glucose. The reduction of the metal ion can result from interaction with any reducing substance present in the urine, such as creatinine, uric acid, ascorbic acid, or some other reducing sugar. The nonspecificity of the copper reduction assay is advantageous in that the assay detects sugars in the urine other than glucose. However, the assay has a disadvantage in that it detects other reducing substances in addition to sugars. ##STR2##
Some dry phase test strips used to assay for glucose have a single test area consisting of a small square pad of a carrier matrix impregnated with glucose oxidase, peroxidase and an oxidizable chromogenic indicator. Other dry phase test strips are multideterminant reagent strips that include one test area for the glucose assay as described above, and further include several additional test areas on the same strip to permit the simultaneous assay of other urinary constituents. For both types of colorimetric dry phase test strips, the assay for glucose in urine is performed simply by dipping the test strip into a well mixed, uncentrifuged urine sample, removing the excess urine by touching the side of the container, then comparing the resulting color of the test area of the test strip to a standardized color chart provided on the colorimetric test strip bottle. For example, in the copper reduction assay used to assay for the presence or concentration of glucose in a liquid test sample, the color resulting from the reaction with glucose is compared to a six-block color chart ranging from blue, indicating less than 0.1% concentration of glucose, to brown, indicating a 2.0% or greater glucose concentration. In accordance with the above-described method, an individual can readily determine, visually, the glucose content of a urine sample in the range of from about 0% to about 2%.
The most significant advantages of the present-day test strips are low cost, ease of use, relatively accurate assays and a short response time. These advantages make test strips ideal for the detection or measurement of glucose in urine by an individual at home. However, although a combination of glucose oxidase, peroxidase, and an oxidizable chromogenic indicator have been used extensively in glucose assays, several problems and disadvantages still exist in glucose assay methods utilizing enzymes. A continuing and significant problem troubling both the manufacturer and the user of the enzymatic test strip is the thermal instability of the enzymatic assay composition during the manufacture, shipment, storage and use of the dry phase test strip.
In order to determine the extent of the stability problem in regard to a particular enzyme composition, manufacturers routinely conduct stability studies at elevated temperatures. The results of these stability studies are used to predict the shelf life, and therefore the expiration date, of the dry phase test strip. Obviously, too short of a shelf life severely limits the viability of a test strip as a commercial product.
As discussed above, glucose test devices are used extensively both by trained medical personnel and by individuals in their own homes. Whereas the storage conditions in hospitals and clinical laboratories are usually relatively well controlled, storage conditions for home users can be extremely variable. In addition, home users may use several strips in a day and therefore consume a bottle of test strips in a short period, or they may use a test strip only relatively infrequently. Of course, the test strips also may be carried with the user and therefore be subject to a wide range of temperature conditions. This is particularly true of the glucose test strips that are used frequently by diabetics. However, the method and composition of the present invention provides improved thermal stability for enzymatic glucose assay compositions, for test strips utilizing a glucose oxidase/peroxidase composition, and for an enzymatic assay composition for other analytes, such that the dry phase test strip is more tolerant of varying manufacturing and storage conditions.
The stability of the test strip is a significant aspect in the commercial use of enzymatic test strips. An enzymatic test strip having a long shelf life can remain in distribution channels, such as in a manufacturer's and distributor's inventory, for long periods before shipment to a laboratory, and the laboratory can order a sufficiently large inventory of test strips so that frequent reordering is not necessary. Furthermore, increasingly critical cost factors are somewhat alleviated because fewer enzymatic test strips would have to be discarded after reaching an expiration date that is based on the shelf life stability of the test strips. As a result, it is important to provide a stable enzymatic test strip that a laboratory or home user will not have to return or discard before use.
Therefore, it would be extremely advantageous to have a simple, accurate and trustworthy method of assaying of urine or serum for glucose content without the disadvantage of unduly short test device shelf life. Such advantages are most evident in situations wherein an out-dated test device was used to assay urine for glucose and the instability of the enzyme reagent composition incorporated into the test device provided erroneous assay results, and hence to an erroneous diagnosis. By providing a stable dry phase test strip for the determination of the glucose concentration in urine or serum, in an easy to use form, such as a dip-and-read test strip, the urine or serum assay for glucose can be performed by laboratory personnel or by an individual at home to more precisely monitor the levels of glucose in urine or serum and/or the success of the medical treatment the individual is undergoing without the disadvantage of possibly using an out-dated and unreliable test strip.
As will be described more fully hereinafter, the method of the present invention allows the fast, accurate and trustworthy assay of urine or serum for glucose by utilizing a dry phase test strip that includes a stabilized enzyme-based reagent composition. The stabilized enzyme reagent composition improves the heat stability and shelf life of the dry phase test strip, thereby allowing the glucose concentration of test samples to be accurately determined using enzymatic test strips that either were manufactured in the relatively distant past or were stored at abnormally high temperatures.
As described previously, glycosuria is related to the precise nature of a clinical and pathological disorder and upon the severity of the specific disease. Glycosuria can be intermittent or continuous, with transient, intermittent glycosuria usually being caused by physiologic or functional conditions rather than by pathologic disorders. Therefore, accurate and trustworthy assays of urine and other test samples for glucose must be available for both laboratory and home use. The assays must permit the detection and measurement of glucose, such that a correct diagnosis can be made and correct medical treatment implemented, monitored and maintained.
Furthermore, any method of assaying for glucose in urine or other liquid test samples must yield accurate, trustworthy and reproducible results by utilizing a stable enzyme reagent composition that undergoes a color transition as a result of, and that can be correlated to, an interaction with the glucose content of the test sample. It is imperative that the assay reflects the true glucose concentration of the test sample and not an apparent, lower glucose concentration as a result of a reduction in the strength of the enzyme reagent composition over time. Additionally, the method and composition utilized in the assay for glucose should not adversely affect or interfere with the other test reagent pads that are present on multiple test pad strips.
Prior to the present invention, no known method of assaying urine or other liquid test samples for glucose included a stabilized enzyme reagent composition that so unexpectedly improved the stability of the dry phase test strip, thereby allowing accurate and trustworthy glucose assays to be made with relatively old and/or improperly stored test strips. In addition, although enzyme-based dry phase test strips have been used extensively in glucose assays for several years, no test strip has incorporated the enzyme reagent composition stabilizers utilized in the present invention to improve the stability of enzyme-based dry phase test strips.
The prior art contains numerous references relating to attempts to stabilize enzyme compositions incorporated into dry phase test strips. For example, Gruber et al, in U.S. Pat. No. 3,721,607, disclosed a stabilized test reagent for the enzymatic determination of glucose that includes both an inorganic azide and the compound 2,2'-azino-di-(3-ethylbenzothiazoline-6-sulfonic acid). However, the compositions disclosed by Gruber are stabilized solutions used in wet enzymatic assays for glucose and are not directed to stabilizing dry phase test strips used in an enzymatic assay for glucose. Similarly, Japanese Patent No. 55/013008 disclosed using lower alcohols to preserve glucose oxidase and/or peroxidase solutions.
European Patent No. 080,304 disclosed the use of an acidic amino acid to stabilize a reagent solution including an enzyme. Acidic amino acids, such as aminodicarboxylic acids and their ammonium and alkali metal salts, like glutamic acid, aspartic acid and o-aminoadipic acid, were used to stabilize an aqueous solution including enzyme, like glucose oxidase and choline oxidase. The European patent does not teach or suggest using any other amino acids or amino acid salts except for the acidic amino acids, and does not teach or suggest using the acidic amino acids as a stabilizer in enzymatic dry phase test strips.
U.S. Pat. No. 3,778,384 to Dooley disclosed the use of an arsenic compound in a glucose assay based on a glucose oxidase/peroxidase reagent composition. The arsenic compound, such as sodium arsenate, increases the sensitivity of enzyme reagent composition to glucose and increases the stability of the color transition occurring as a result of the glucose-enzyme interaction. Dooley also disclosed including thiourea in the composition to further stabilize the color transition. In contrast, the present invention is directed to stabilizing the enzyme reagent composition such that after prolonged storage, even under adverse storage conditions, the reagent composition has maintained its enzymatic and chemical activity and, therefore, the assay for glucose in urine or serum provides accurate and trustworthy results.
Green, in U.S. Pat. No. 4,189,536, disclosed using the tris(hydroxymethyl)aminomethane salt of 2-mercaptosuccinic acid as an activator or stabilizer in assays utilizing enzymes, like assays for creatine phosphokinase or glucose. However, similar to the compositions disclosed by Gruber in U.S. Pat. No. 3,721,607, the compounds disclosed by Green serve to stabilize the enzyme reagent first in the bulky dry storage phase and, then in the aqueous phase, after dissolution of the enzyme reagent, for improved performance of wet phase chemical assays. The compound disclosed by Green is used as a bulking agent and stabilizer for reagent compositions based upon the hexokinase catalyzed phosphorylation of glucose and the subsequent oxidation of glucose-6-phosphate, as opposed to an enzyme reagent stabilizer for the dry phase test strip assay of glucose based upon glucose oxidase and a peroxidase.
U.S. Pat. No. 4,220,713 to Rittersdorf et al disclosed the use of 1-arylsemicarbazides as stabilizers in test strips for colorimetric assays utilizing an oxidizable indicator, such as a glucose assay based on glucose oxidase and peroxidase. The parent compound, semicarbazide, specifically is described as being nonfunctional as a stabilizer. In contrast, the method and composition of the present invention is directed to various hydrazides and to certain amino acids and amino acid derivatives that stabilize enzymatic test strips used to assay for analytes.
Motobayashi, in U.S. Pat. No. 4,386,053, disclosed a stabilizer for an occult blood assay device that incorporates an organic peroxide, a chromogen and a buffer. The stabilizer is an aromatic compound having a substituted phenyl moiety and a substituted amide or substituted sulfonamide moiety. Similarly, European Patent No. 071,730 to Klose disclosed a stabilized reagent mixture for the detection of hydrogen peroxide or hydrogen peroxide-forming systems including methylbenzothiazolone hydrazone, an oxidizable aromatic amine and a ferrocyanide, azide or chelating agent as a stabilizer.
In contrast to the prior art, and in contrast to presently available commercial test strips, the method and composition of the present invention provides increased stability to dry phase enzymatic test strips used in the detection and measurement of glucose and other analytes in urine and serum by utilizing a hydrazide stabilizer or an amino acid stabilizer or an amino acid derivative stabilizer. Unexpectedly improved stability of the enzyme reagent composition results when both a hydrazide and an amino acid, or both a hydrazide and an amino acid derivative, is utilized as the stabilizer of the enzyme reagent composition. Hence, in accordance with the method and composition of the present invention, new and unexpected results are achieved in stabilizing dry phase enzymatic test strips used to assay urine and other liquid test samples for analytes, such as glucose and cholesterol, by incorporating a hydrazide stabilized or an amino acid stabilized or an amino acid derivative stabilized enzyme reagent composition into a carrier matrix.